Sara Pratt and Dr. Daniel Austin, Department of Chemistry and Biochemistry
In this project we wanted to see if bacteria can survive a process called electrospray ionization (ESI). While bacteria survival of ESI has not previously been demonstrated, viruses survivability has1. The process of ESI consists of spraying a solution of the desired analyte in a strong electric field. Solution flowing through a capillary forms a Taylor cone at the end from which charged droplets emit. As solvent evaporates, the droplets break apart. As solvent continues to evaporate, that process is repeated and eventually forms individually charged particles once all the solvent is evaporated. The ESI in this experiment is done with a positive bias.
Our apparatus consists of an electrospray source which sprays through an ion mobility drift tube toward a collection plate. For the experiments, 5000 V is applied to needle and 3500 V is applied to the resistive tube. The collection plate is grounded so that the bacteria are attracted to it. A picoammeter is connected to the collection plate to monitor the current of the spray hitting it. N2 can be flowed through the gas inlet to exclude neutral particles. Both horizontal and vertical configurations of our apparatus have been tested.
One reason why we wanted to know if bacteria can survive ESI is that we have another apparatus that can accelerate particles which are electrosprayed into it. If bacteria can survive electrospray then we can use that other apparatus to see what kind of impacts they can survive. NASA is interested in the results of those tests because they want to know if bacteria can survive impacts such as spacecraft crashes to evaluate the potential for contamination of other planetary bodies during space exploration. Other work2 has been done, but that has focused on survivability in the interior of meteorites.
For our tests, we started using ampicillin-resistant E. coli, which were cultured in a liquid media. From the liquid media, the bacteria are diluted ten fold in pH 7 buffer and filtered through glass wool to eliminate aggregates. Thus, were bacteria surviving are not aggregated. Before spraying bacteria onto the collection plate, it was sterilized with methanol. After E. coli were electrosprayed onto the collection plate, it is swabbed and any sprayed bacteria were cultured in an ampicillin-containing media. Thus we know that the bacteria that- grew were the ones we sprayed in. The collection plate was sterilized before each experiment.
From these experiments we have found that the E. coli are able to survive the charging process of electrospray, but not the desolvation process. When we sprayed directly onto the collection plate without the drift tube in between, there was still liquid left and the E. coli culture results were consistently positive. When they were sprayed vertically down the tube, we saw droplets on the collection plate and the bacteria survived. When we sprayed them horizontally we did not see water droplets, and the bacteria did not survive. When we built a new electrospray source that used a flow of nitrogen around the needle as a nebulizing gas to further desolvate the bacteria, they did not survive vertical electrospray. These results lead us to believe that they are surviving being charged, but not losing their solvent. I presented the E. coli results at both the Spring Research Conference at BYU and the National ACS Meeting in Anaheim.
We are currently in the process of experimenting with Bacillus atrophaeus endospores. Bacteria form endospores when confronted with nutrient depravation. These spores can survive harsh conditions including dryness, so we hypothesized that they could survive both the charging and desolvation of ESI. So far, the tests indicate that they can. They have survived horizontal tests with both a nebulizing gas that further desolvates them and a counter gas that would exclude non-charged particles. These results indicate that they are both charged and desolvated when they hit the collection plate. We have a few other strains of spore forming bacteria that we will also test. Now that we have demonstrated ESI survival, the next step is to use the accelerating instrument to see what kind of impacts they can survive.
References
- Siuzdak, Gary; Bothner, Brian; Yeager, Mark; Brugidou, Christophe; Fauquet, Claude M.; Hoey, Kenway; Chang, Cheng Ming. Mass spectrometry and viral analysis. Chemistry & Biology. 1996, Vol 3 No 1, 45-48.
- Horneck, G; Stoffler, D.; Eschweiller, U.; Hornemann, I. Bacterial spores survive simulated meteorite impact Icarus. 2001, 149, 285-290.